Abstract

In order to address how plant-fungal interactions in natural soil shape and are shaped by local and systemic responses to beneficial and pathogenic root associated fungi a reductionist approach which takes advantage of a gnotobiotic natural soil-based split root system was established.

Phenotyping, cytological and transcriptional analyses during barley infection with the root rot fungal pathogen Bipolaris sorokiniana, and colonization with the beneficial root endophyte Serendipita vermifera showed remarkably distinct responses of the host. Whereas the root endophyte only marginally affected the expression of plant genes, 2741 host genes were deregulated by pathogen infection. The presence of the root endophyte significantly reduced pathogen infection and disease symptoms increasing host resistance rather than tolerance against the pathogen without markedly altering plant response at the transcriptional level.

The pathogenic fungus utilized secondary metabolites to antagonize the beneficial fungus whereas the root endophyte employed proteins with hydrolytic activities to parasitize the other fungus, including a chitinase with a CBM5-12 carbohydrate-binding domain found specifically in Agaricomycotina and chitinolitic bacteria. The identified sebacinoid mycoparasitic genes from soil confrontations are not induced in planta during tripartite interaction, suggesting that the two fungi are not in direct contact inside the root and niche differentiation might occur.

Systemic and local plant responses indicate that a plant component is involved in the increased resistance to the pathogen other than priming.

Abstract

Roots and their surfaces host diverse microbial communities. These rhizobiomes are currently attracting vast interest as a resource for designing microbial consortia that could be harnessed to improve restoration and revegetation success, and to improve yields while minimizing inputs in agricultural systems. Such efforts seem justified as the rhizobiomes are intimately associated with plant tissues, have the potential to regulate plant performance, and may be able to modify the host phenotypes and environmental tolerances. However, before such designed, synthetic consortia can be exploited, research to characterize the communities and – particularly – their stable or core members is mandatory. In the course of an ongoing collaboration, we investigated the biogeography of the fungal rhizobiomes associated with foundation grasses (Andropogon gerardii (big bluestem), Bouteloua eriopoda (black grama), B. gracilis (blue grama), B.dactyloides (buffalo grass), and Schizachyrium scoparium (little bluestem) across latitudinal gradients within the North American Great Plains. Our primary goal was to gain a deeper understanding of the relative importance and ranking of climatic, edaphic, geographic and host traits on the diversity and composition of the fungal rhizobiome. To do this, we sampled twelve individuals of each of the five grass species from 24 sites spanning across a N-S and W-E gradients in south and south central US. Our data indicate a host grass species effect amongst the grasses, and a distinct latitudinal interaction as driver for the fungal rhizobiome. In terms of the environmental drivers, pH tended to be the strongest predictor, highlighting the edaphic controls of rhizobiomes. This study improves our understanding of the compositional drivers of fungal rhizobiomes associated with these foundation grasses, permitting a better prediction of compositional community shifts in changing environments.

Abstract

The exposure of fresh rock surfaces and primary minerals in response to landsliding sets in motion biogeochemical and biological processes that have not been conceptually linked. On the one hand landslide activity may enhance chemical weathering and the release of mineral-bound nutrients which may explain why landslide activity is associated with an increase of some mineral-derived nutrients. On the other hand, microbial communities, especially fungi associated with plant roots play a major role in rock weathering. Of particular interest is the weathering of (Ca, Mg)-bearing silicate rocks because this process plays a critical role in the long-term carbon cycle. The main goal of this study is to characterize the rhizobiomes of plants developing in contrasting weathering environments, namely anthropogenic landslides and forest underlain by carbon and magnesium (Ca, Mg)-bearing silicate rocks in a tropical environment. We collected bulk soil and soil from roots of eleven plant species belonging to three life forms (terrestrial fern, arboreal fern, and shrubs) growing at landslide and forest sites underlain by granodiorite of the Utuado batholith in central Puerto Rico. We extracted DNA from these soil samples using the MoBio Powersoil kit and sequenced the the ITS (fungal) region in the MiSeq platform. We expect that species richness and composition of fungal communities will differ between habitats but not among life forms. Specifically, we expect higher fungal species richness in landslides than forests. To our knowledge, this is the first microbiome study using Next-Generation Sequencing (NGS) technologies to link fungal rhizobiota to the short and long-term carbon cycles in areas underlain by silicate rocks subjected to landslide activity.

Abstract

Plants are associated with a suite of microbial symbioses, with roots offering a unique niche for fungal endophytes. Among root fungal symbionts, dark septate endophytic (DSE) fungi are common, sometimes abundant but enigmatic with poor clarity on their functional roles. Biogeographical distinctions likely exist in DSE communities from forests and grasslands, with North American and European grasslands predominantly represented by Periconia macrospinosa. To understand their endophytic roles, recently the genome of dark septate P. macrospinosa and Cadophora isolated from Festuca vaginata from semi-arid European grassland were sequenced. To further comprehend DSE functional roles, the objectives of this study were to i) compare the North American P. macrospinosa genome with that of the European P. macrospinosa; ii) gain insights into P. macrospinosa global proteome profiles; and iii) gain insights into Periconia transcriptomics under grass symbiosis. Periconia was isolated from a stand of Freedom Giant Miscanthus cultivated in Lorman, Mississippi and was confirmed to be a DSE. We hypothesized that despite the geographical distinctions and diverse grass hosts, P. macrospinosa associated with grasses would have similar functional roles. Periconia genome was sequenced using Illumina and PacBio platforms. Our Periconia genome was determined to be ~ 53.5 MB in size with 45% GC content. At least 12,059 ORFs with 9,086 ORFs with introns were identified and nearly 35% of the ORFs were assigned functions. As expected, several plant cell wall degrading enzymes (PCWDEs) like cellulases (12 ORFs), amylases (2 ORFs), pectin esterase (1 ORF), tannase (2 ORFs), laccase (6 ORFs) were identified along with several sugar transport systems such as maltose, lactate, sucrose, maltose, xylose isomaltose, palatinose, etc. However, ORFs for lignin peroxidase, manganese peroxidase, glyoxal oxidase were not observed. For global proteomic profiling, label free quantitative (LFQ) profiling using UPLC-MS/MS was used. For DSE gene expression insights, transcriptomic studies were performed on symbiotic-Periconia ten days post inoculation with Miscanthus sinensis. Periconia macrospinosa genomics, expression and proteomes data will be discussed to draw big picture inferences regarding DSE symbiosis.

Abstract

The temperate rainforests of Western Washington are known for their temporally stable old-growth forests and unique ecosystem processes, including seasonal rainfall regimes. In recent years, they have been experiencing more seasonal extremes, including wetter winters and drier summers. Canopy soils, which form on tree branches from mossy epiphytic mats intercepting literfall and decomposing over time, are prevalent throughout these ecosystems. Some of the old-growth tree species have adapted to the presence of canopy soils by growing adventitious roots into this organic soil horizon. The aim of this research is to elucidate the role of fungi associating with adventitious roots in old-growth Acer macrophylum trees. Preliminary DNA and microscopy/imaging analyses suggest that these adventitious roots have adapted to form a diversity of fungal root associates that differ from those found in the forest floor rooting networks. Soil microclimatic and nutrient data suggests that canopy and forest floor characteristics are significantly different (p<.05), and there are hotspots of available P and N in the canopy soil environment. Soil microclimate and available nutrients could both be factors influencing fungal community structure. Preliminary DNA analysis was performed by cloning and Sanger sequencing, and although highly accurate, only some inferences on the identity of fungi associating with canopy roots could be made. Due to the cryptic nature of fungi and the paucity of information regarding fungi in canopy soils, a protocol was created to approach fungal community analysis using Oxford’s high-throughput MinIon Nanopore Sequencer. The MinIon approach was selected based on the the technology’s potential to provide long-read barcoded libraries (~1,500 bp). DNA was extracted from adventitious and forest floor root-tips of six A. macrophylum trees from two old-growth forest stands, using OPS Diagnostics Synergy 2.0 Plant Extraction Kit. Based on the ability to amplify species from Ascomycota, Basidiomycota, and Glomeromycota, primers ITS1F-KYO and a variant of LR3, with custom tails respective to Oxford’s 1D barcoding protocol, were selected for PCR. Following PCR, individual root-tips were assigned a unique barcode, pooled together, and the barcoded library was loaded into the MinIon for sequencing. The MinIon Sequencer ran overnight, returning ~800,000 fungal sequences. Currently, we are working on an efficient workflow to filter, process, and analyze the entire library of long-read sequences. Subsets of this data form a healthy amount of OTUs from Ascomycota, Basidiomycota, and Glomeromycota, and return identities that match as high as 95% in the NCBI and UNITE databases. Results from this fungal community analysis, and the potential capability of the MinIon Nanopore Sequencer being a sufficient approach to fungal community analysis will be reported.

Abstract

Root-endophytic fungi are ubiquitous and abundant in terrestrial plants, likely playing roles in plant health and productivity. They are frequently considered key drivers of ecosystems' function and promising tools for agriculture, but important knowledge gaps on their ecology limit the understanding of their place in ecosystems, as well as their exploitation. Fungal endophytes are phylogenetically and functionally diverse, therefore, unraveling their trophic lifestyles, their impacts on plant fitness, and their taxonomy may help infer their various ecological roles. We sought to gain insight into the evolution and functional diversity of these fungi through a comparative study of their traits, and by providing quantitative linkages between sets of traits and endophytes’ community ecology. Using both high-throughput sequencing and cultivation methods, we assessed the fungal diversity within roots of non-mycorrhizal plants across Europe to evaluate differences in host-dependency and distribution between cultivable and non-cultivable endophytes. The samplings enabled the assembly of a collection of ca. 2,500 isolates, representing the majority of dominant endophytic lineages, and their subsequent characterization using multilocus genotyping; measurements of morphological, physiological, and chemical traits; and bioassays with different hosts to determine their impacts on plant growth. We found a consistent dominance of root-endophytic communities by few cultivable fungal taxa, implying that facultative plant associations are pervasive in the core fungome of non-mycorrhizal roots. Dominant fungal lineages displayed different ecological preferences and complementary sets of traits, suggesting niche partitioning as an important driver of the assembly of root-endophytic communities. Experiments in which roots were co-inoculated with representative isolates supported this hypothesis by showing little interaction between endophytes. In conclusion, root-endophytic fungi are not functionally homogeneous, but instead form communities where a partitioned exploitation of niches takes place. Our findings enable to postulate hypotheses about the potential ecological roles of different endophytic groups, although they are not specific in attributing particular functions. Further approaches to assess the implication of fungi in specific symbiotic processes will be discussed.